Rabbit polyclonal anti β actin

Western blotting was performed as described previously (Dong et al., 2011 (link)). Briefly, NCCs were washed with phosphate-buffered saline (PBS) and then lysed in pre-cold RIPA lysis buffer (Cell Signaling, Beverly, MA) with 1 mM fresh-prepared PMSF (Sigma-Aldrich, St Louis, MO) and protease cocktail inhibitors (Roche Applied, Indianapolis, IN). Cell lysates were then centrifuged at 12,000 xg for 10 min at 4°C and the supernatants were used for Western blot. The protein concentration in each sample was determined using BCA protein assay kit (Pierce, Rockford, IL) following the manufacturer’s instructions. Western blots were performed by standard protocols. The levels of Bak1, PUMA, caspase-3 and β-actin were analyzed with the following antibodies, respectively: rabbit polyclonal anti-Bak antibody (Cell Signaling, Beverly, MA), rabbit polyclonal anti-PUMA antibody (Abcam, Cambridge, MA), rabbit monoclonal anti-cleaved caspase-3 antibody (Cell Signaling, Beverly, MA), and rabbit polyclonal anti-β-actin (Santa Cruz, Santa Cruz, CA). The membranes were developed on a Kodak X-OMAT 2000A imaging system (Kodak, Rochester, NY) and the intensity of the protein band was analyzed using the Adobe Photoshop CS software (Adobe Systems, San Jose, CA).